Monoclonal antibodies for the direct detection of influenza-A virus by ELISA in clinical specimens from patients with respiratory infections

Abstract

Background: Monoclonal antibody technology provides antibody reagents of known specificity, high titres and unlimited availability, that form ideal reference antibodies for use in specific viral antigen-detection methods. Objectives: To produce mouse monoclonal antibodies against antigenic sites of influenza-A virus, and evaluate their use as diagnostic reagents in a sandwich ELISA. Study design: (1) Production and characterization of monoclonal antibodies against influenza-A virus; (2) application of these antibodies in an ELISA method for direct antigen detection; and (3) evaluation of the ELISA as routine procedure. Results: Four monoclonal antibodies (A1–A4) from mice immunized intranasally with influenza-A virus were selected according to their specific reactivity with either nucleoprotein or matrix protein antigens as demonstrated by Western blot analysis. These antibodies lacked haemagglutination inhibition and neutralization properties and recognized both H1N1 and H3N2 strains of influenza-A virus equally. A sandwich ELISA using unlabelled antibodies for antigen capture and biotin-labelled antibodies for antigen detection was used to analyse nasopharyngeal secretions or nasal swabs from culture-confirmed influenza-A-infected patients and comparable specimens from patients with other viral respiratory infections. Only influenza-A virus (strains H1N1 and H3N2) could be detected in samples from patients with known influenza-A and influenza-B infections, and also after re-isolation of such viruses in conventional cultures of MDCK cells or embryonated hens' eggs. The antigen-detection assay showed a diagnostic sensitivity of 100% and a specificity of 98.3% compared with conventional culture methods. Conclusion: The reported ELISA appears to be a rapid and inexpensive method for diagnosis and epidemiological studies of influenza-A infections.